When a target foreign gene is introduced into and expressed in a plant, a constitutive promoter that is expressed at all times, such as a cauliflower mosaic virus (CaMV) 35S promoter (e.g., Benfey P N & Chua N H, 1990, Science 250, 959-966) is often used. However, in the case of using such a constitutive promoter, a heavy burden is posed on transcription and translation systems, and depending on a kind of a target foreign gene, adverse effects such as inhibition of germination and growth may be sometimes given to a plant. As a method of avoiding such adverse effects, a method of inducing and expressing a target foreign gene at a desired time can be mentioned. By utilizing such an induced expression method, it becomes possible to express a target foreign gene more efficiently, thereby to realize enhanced production of a target foreign protein, etc., encoded by a structural gene region contained in the foreign gene, to control growth and physiology of a plant freely, whose industrial utility value is invaluable.
Methods for induced expression of a target foreign gene at a desired time can be broadly classified into two methods according to the difference in inducing conditions. Namely, there are a method for expression induced by a non-chemical substance such as temperature, light, or a plant pathogen; and a method for expression induced by a chemical substance.
Examples of the former method include a heat inducible system (e.g., U.S. Pat. No. 5,447,858), a low-temperature inducible system (e.g., U.S. Pat. No. 5,847,102) and a system induced by attack of a plant pathogen (e.g., U.S. Pat. No. 5,942,662). These systems utilize only a gene construct having such a very simple structure that an inducible promoter is linked to an upstream region from a target foreign gene. However, in the case of expression induced by a non-chemical substance such as temperature, light, or a plant pathogen, there is a risk that a target foreign gene is abruptly expressed by an unexpected change of an environment, attack of a plant pathogen, or the like.
On the other hand, examples of the latter method include a copper ion-inducible system (e.g., Mett V L et al, 1993, Proc Natl Acad Sci 90, 4567-4571), a steroid hormone-inducible system (e.g., Aoyama T & Chua N H, 1997, Plant J. 11, 605-612; U.S. Pat. No. 6,063,985), an ethanol-inducible system (e.g., Caddick M X et al, 1998, Nature Biotech. 16, 177-180; WO 93/21334) and a tetracycline-inducible system (e.g., Weinmann P et al, 1994, Plant J. 5, 559-569). These have been explained in detail by Moore I et al (Moore I et al, 2006, Plant J 45, 651-683) and Padidam M (Padidam M, 2003, Current Opin Plant Biol 6, 169-177), and the expression of a target foreign gene can be controlled depending on the concentration of a specific chemical substance. Namely, the expression of a target foreign gene can be induced to a necessary amount at a desired time.
Conventionally, in order to produce a target foreign protein that is encoded by a structural gene region contained in a target foreign gene in a recombinant plant (accumulation in the plant), there are some methods which utilize an Ω sequence of a tobacco mosaic virus (TMV) (e.g., Gallie D et al, 1987, Nucleic Acid Res 15, 3257-3273; U.S. Pat. No. 5,489,527) or a 5′-untranslated region sequence of a tobacco alcohol dehydrogenase gene (e.g., Satoh J et al, 2004, J Biosci Bioeng 98(1):1-8; JP 2003-079372 A) to be located upstream from a structural gene region contained in a target foreign gene. On the other hand, there is a report showing that a mRNA linked to an Ω sequence is easily degraded by a nuclease (e.g., Gallie D et al, 1988, Nucleic Acid Res 16, 8675-8694). Then, what kind of results is brought by utilizing a 5′-untranslated region sequence cannot be readily presumed. Besides, it has been totally unknown what kind of influence is brought on expression of a target foreign gene induced by a chemical substance in a specific inducible system (for example, a copper ion-inducible system, etc.)